Summary RNAsynthesistechnologieshavebecomecriticalinbasicbiomedicalresearch,andindevelopmentofRNA?based sensors,diagnosticsandtherapeutics1?6.Currently,RNAsaresynthesizedbyeithersolid?phasephosphoramidite chemistry7,8,orbytranscriptioninsolutionwithphage?encodedRNApolymerases10.Thestructuraldiversityof chemicallysynthesizedRNAsislimitedbytheavailabilityofthephosphoramiditesandthecompatibilityofreaction chemistrywithdifferentnucleotidemodifications,butthemoreimportantlimitationofchemicalsynthesisisthatRNAs longerthanafewtensofnucleotidescannotbemadewithacceptableyields11.Enzymaticmethodsallowsynthesisof muchlongerRNAs12,andbyusingspeciallyengineeredRNAPs13?18,evenallowsynthesisofRNAswithnon?canonical chemistrytoenhancefunctionalityorRNAseresistance19,20,butalimitationofenzymaticsynthesisisthatthechemical compositionofanRNAishomogeneouslydeterminedbythemixofNTPsinthereaction(i.e.,differentsegmentsofone RNAcannothavedifferentchemistry).Werecentlypublishedaproof?of?principlestudyforanewRNAsynthesis technology??PLOR(forpositionallabelingofRNA)??thatcombinessolid?phaseandenzymaticsynthesistoallow preparationofindefinitelylongRNAsinwhichmultiplespecificsegmentsornucleotidescanbespecificallylabeledwith distinctchemistry21.ThistechnologyrepresentsaquantumleapinourabilitytocharacterizeRNAstructureand mechanism,toprepareRNAaptamersorinterferingRNAsspecificallyderivatizedtooptimizetheirdelivery,stabilityor effectivenessinvivo,andalsopresentspotentialadvantagesinefficiencythatmaysupersedecurrentsynthesismethods evenforconventional(singlechemistry)RNAs.Theproposedworkwilloptimizetheefficiencyandeconomicsofthis noveltechnology,expanditsutilityforsynthesisofRNAswithnon?canonicalnucleotidechemistries,and,ingeneral, makepossibletheroutineandeconomicallyefficientpreparationofanewgenerationofmosaic,chemicallydiverseRNA moleculeswithapplicationsrangingfromfundamentalbiomedicalresearchtoclinicaltherapeutics. 1